Inhibition by docosahexaenoic acid of receptor-mediated Ca(2+) influx in rat vascular smooth muscle cells stimulated with 5-hydroxytryptamine

C-type natriuretic peptide (CNP) has vasodilatory and antimitogenic actions, but its role in the control of cardiac function is unclear. We studied the effect of CNP on cultured, beating neonatal rat cardiac myocytes. CNP caused a significant reduction in the amplitude of contraction and a significant accumulation of intracellular cyclic GMP. The effect of a membrane permeable cyclic GMP on cell contraction was similar to that of CNP. CNP caused no change in Ca2+ transients. Blockade of natriuretic peptide receptors abolished the effects of CNP on contraction and accumulation of intracellular cyclic GMP. Blockade of cyclic GMP-dependent protein kinase abolished the effect of CNP on myocyte contraction. We conclude that CNP has a negative inotropic effect on neonatal rat cardiac myocytes. The effect of CNP is mediated via natriuretic peptide receptor(s) causing elevation of intracellular cyclic GMP which possibly activates protein kinase and causes attenuation of myofilament sensitivity to Ca2+.     

Effect of alpha-human atrial natriuretic peptide on the synthesis of dopamine in the rat kidney.

1. The present study has examined the influence of alpha-human atrial natriuretic peptide (alpha-hANP) on the synthesis of dopamine and its deamination into 3,4-dihydroxyphenylacetic acid (DOPAC) in rat kidney slices loaded with exogenous L-dihydroxyphenylalanine (L-DOPA). 2. alpha-hANP (3.3 and 330 nM) was found to produce a marked reduction (63-78% reduction) in the time-dependent accumulation of newly-formed dopamine and of its deaminated metabolite DOPAC in kidney slices loaded with 10 microM L-DOPA. alpha-hANP (330 nM) was also found to decrease the accumulation of newly-formed dopamine (45-66% reduction) and DOPAC (38-61% reduction) in experiments in which increasing concentrations (1-100 microM) of L-DOPA were used. This inhibitory effect was found to be potentiated by zaprinast (M&B 22,948; 10 microM), a guanosine cyclic 3',5'-monophosphate (cyclic GMP) phosphodiesterase inhibitor. Alone, zaprinast also decreased the accumulation of both dopamine (54-71% reduction) and DOPAC (73-92% reduction). 3. In kidney homogenates, alpha-hANP (330 nM) was found to affect neither the formation of dopamine nor its deamination to DOPAC. 4. Both alpha-hANP (330 nM) and zaprinast (10 microM) were found not to affect the formation of dopamine and DOPAC in kidney slices obtained from rats on a high salt diet during the previous 6 weeks. A similar situation was also found to occur when kidney slices obtained from 24-months old rats were used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide donors enhance calcitonin gene-related peptide-induced elevations of cyclic AMP in vascular smooth muscle cells.

Vasorelaxant effects of calcitonin gene-related peptide (CGRP) are dependent on endothelium-derived nitric oxide (NO) in some arteries. The mechanism involved is still not clear. In the present study, we used NO donors (sodium nitroprusside (SNP) and 6-(2-hydroxy-1-methyl-2-nitrisohydrazino)-N-methyl-1-hyxanamine (NOC-9)), cyclic GMP elevator (brain natriuretic peptide (BNP)) and a selective type III (cyclic GMP-inhibited) phosphodiesterase (PDE) inhibitor 5-(4-acetamidophenyl)pyrazin-2(1H)-one (SK&F94120) to investigate involvement of NO, cyclic GMP and type III PDE in CGRP-induced accumulation of cyclic AMP in cultured rat aortic smooth muscle cells. SNP (10 microM), NOC-9 (10 microM) and BNP (1 microM) all increased intracellular cyclic GMP to similar levels (2- to 2.5-fold above basal) and caused significant enhancement of CGRP (10 nM)-induced cyclic AMP accumulation similar to that caused by 10 microM SK&F 94120. The data are therefore consistent with our hypothesis that the mechanism of endothelium-dependent vasorelaxation effect of CGRP involves cyclic GMP-mediated inhibition of type III PDE and subsequent accumulation of cyclic AMP in smooth muscle cells.     

Stimulatory effects of brain natriuretic peptide on cyclic GMP accumulation and tyrosine hydroxylase activity in cultured bovine adrenal medullary cells

Summary We studied the effect of brain natriuretic peptide (BNP) on the accumulation of cyclic GMP and the phosphorylation and activity of tyrosine hydroxylase, compared with that of atrial natriuretic peptide (ANP), in cultured bovine adrenal medullary cells. 1. BNP as well as ANP increased cellular cyclic GMP accumulation in a concentration-dependent manner (10–1000 nmol/1). BNP (1 mol/1) and ANP (1 mol/1) produced a 60-fold and 30-fold increase in cyclic GMP accumulation, respectively. 2. The stimulatory effects of BNP and ANP on cyclic GMP accumulation were observed even when Ca²⁺ or Na⁺ was removed from the incubation medium. 3. 12-O-Tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, inhibited the stimulatory effect of BNP on cyclic GMP accumulation in a concentration-dependent manner (1–100 nmol/1). Furthermore, the BNP-induced accumulation of cyclic GMP was attenuated by forskolin (1 mol/1), an activator of adenylate cyclase. 4. BNP (1 mol/1) and ANP (1 mol/1) caused a significant increase in phosphorylation and activity of tyrosine hydroxylase in the cells. 5. In digitonin-permeabilized cells, cyclic GMP (1–100 mol/1) activated tyrosine hydroxylase in the presence of ATP and Mg²⁺.These results suggest that BNP stimulates the accumulation of cyclic GMP in a manner similar to that of ANP. The increased accumulation of cyclic GMP by these peptides may be negatively modulated by protein kinase C and cyclic AMP and may cause the phosphorylation and activation of tyrosine hydroxylase-in cultured bovine adrenal medullary cells.     

Modulation of atrial natriuretic peptide-induced cGMP accumulation by [Arg8]vasopressin in the cultured renal epithelial cell line, LLC-PK1

Atrial natriuretic peptide (ANP) III stimulated the formation of intracellular cGMP in renal epithelial cells (LLC-PK1) dose dependently. Incubation of LLC-PK1 cells with [Arg8]vasopressin resulted in an increase in intracellular cAMP levels. The stimulatory effect of ANP III on the cyclic cGMP system was inhibited by coincubation with [Arg8]vasopressin in a dose-dependent manner.     

Inhibitory action of alpha-human atrial natriuretic peptide on noradrenaline-induced synthesis of myo-inositol 1,4,5-trisphosphate in the smooth muscle cells of rabbit aorta.

1. Interactions between the synthesis of myo-inositol 1,4,5-trisphosphate (IP3) and guanosine 3':5'-cyclic monophosphate (cyclic GMP) in the smooth muscle cells of the rabbit aorta were investigated. 2. In the presence or absence of vascular endothelium, noradrenaline (NA; 5 microM) consistently reduced the amount of phosphatidylinositol 4,5-bisphosphate (PI-P2) and increased both phosphatidic acid (PA) and IP3. 3. In the presence or absence of endothelium, acetylcholine (ACh; 100 microM but not 5 microM) slightly increased the amount of IP3, but exposure to ACh (100 microM) 4 min after application of NA did not modify NA-induced synthesis of IP3. 4. ACh (100 microM) markedly enhanced the synthesis of cyclic GMP in the presence of endothelium but not in the endothelium-denuded tissues. 5. Prazosin (5 microM) but not dibutyryl cyclic GMP (db-cyclic GMP; 100 microM) blocked the hydrolysis of PI-P2 induced by 5 microM NA. Synthesis of IP3 induced by NA, as estimated with [3H]-inositol was not modified by application of 100 microM db-cyclic AMP or db-cyclic GMP. 6. alpha-Human atrial natriuretic peptide (alpha-hANP; 0.1 microM) increased cyclic GMP in the presence or absence of endothelium. alpha-hANP (0.1 microM) consistently inhibited the hydrolysis of PI-P2 induced by 5 microM NA. 7. The results indicate that synthesis of IP3 is inhibited neither by the synthesis of cyclic GMP in the cytosol nor by cyclic GMP itself. However, synthesis of IP3 through hydrolysis of PI-P2 may be inhibited by an interaction between some steps in the IP3 synthetic process and by the activation of the alpha-hANP-guanylate cyclase process at the sarcolemma.     

Comparative effects of activation of soluble and particulate guanylyl cyclase on cyclic GMP elevation and relaxation of bovine tracheal smooth muscle.

1. The effects of nitric oxide-donating compounds and atrial natriuretic peptide on cyclic GMP accumulation and mechanical tone were compared with the effects of isoprenaline in bovine tracheal smooth muscle. 2. Sodium nitroprusside, glyceryl trinitrate, S-nitroso-N-acetylpenicillamine (SNAP), atrial natriuretic peptide and isoprenaline each caused concentration-dependent inhibitions of histamine-maintained tone (EC50 values 320 +/- 80, 150 +/- 45, 14,000 +/- 4,000, 2.8 +/- 0.8 and 6.6 +/- 4.3 nM respectively). 3. When compared with their effects on histamine-induced tone, sodium nitroprusside was equally potent and effective in causing relaxation of methacholine-supported tone (EC50 290 +/- 90 nM) while isoprenaline was as effective, but less potent (EC50 30 +/- 7 nM). SNAP was more potent and equi-effective as a relaxant of methacholine-supported tone (EC50 340 +/- 140 nM). At the maximum concentrations of glyceryl trinitrate and atrial natriuretic peptide tested against methacholine-supported tone, relaxations of 52% and 14% of the isoprenaline maximum were seen. 4. Sodium nitroprusside, glyceryl trinitrate and atrial natriuretic peptide each induced concentration-dependent increases in cyclic GMP accumulation. The time-courses of accumulation correlated closely with the relaxant actions of these compounds. 5. Pretreatment of tracheal smooth muscle with sodium nitroprusside or SNAP caused a rightward shift of the concentration-effect curve for histamine while reducing the maximum response. 6. LY 83583, a putative guanylyl cyclase inhibitor, caused a concentration-dependent reduction in basal cyclic GMP accumulation in tracheal smooth muscle and inhibited the effects of sodium nitroprusside on cyclic GMP accumulation. 7. LY 83583 also inhibited the relaxation of histamine-supported tone by glyceryl trinitrate, sodium nitroprusside, SNAP and atrial natriuretic peptide, and also sodium nitroprusside- and SNAP-induced relaxation of methacholine-supported tone. However, it had no significant effect on glyceryl trinitrate-induced relaxation of methacholine-supported tone. 8. It is concluded that the relaxant actions of sodium nitroprusside, glyceryl trinitrate, SNAP and atrial natriuretic peptide follow as a result of their ability to activate either soluble or particulate guanylyl cyclase leading to cyclic GMP accumulation. Although there does not seem to be any functional difference in the relaxant response to cyclic GMP generated by the particulate as opposed to soluble form(s) of guanylyl cyclase, atrial natriuretic peptide receptor/guanylyl cyclase activation was much less effective in causing relaxation of methacholine-supported tone when compared to activators of soluble guanylyl cyclase.     

Different effects of ANP and nitroprusside on cyclic GMP extrusion of isolated aorta

Atrial natriuretic peptide (ANP) and sodium nitroprusside have potent vasodilator effects on the noradrenaline-precontracted isolated rabbit aorta. A distinct elevation of cyclic GMP in the aortic tissue was observed after both vasodilators. In contrast to sodium nitroprusside, ANP-(5-28) induced a dose-dependent cyclic GMP extrusion from the tissue into the medium. Thus, release of cyclic GMP appears to be specific for activation of particulate guanylate cyclase and provides a mechanism in addition to synthesis and degradation by which the cells can regulate their internal concentrations of cyclic GMP.     

Selective inhibition by ethanol of glutamate-stimulated cyclic GMP production in primary cultures of cerebellar granule cells

In primary cultures of cerebellar granule cells of the rat, the accumulation of cyclic GMP was stimulated by glutamate, acting at the N-methyl-D-aspartate recognition site, and by atrial natriuretic factor. The response to glutamate was calcium-dependent, while the response to atrial natriuretic factor was not. Ethanol inhibited the accumulation of cyclic GMP in response to both glutamate and atrial natriuretic factor. However, the response to glutamate was much more sensitive to ethanol, with 30-40% inhibition occurring at 50 mM ethanol. Substantial inhibition of the response to atrial natriuretic factor was observed only at concentrations of ethanol of 200 mM or larger. The data suggest that a major site of action of ethanol in inhibiting the accumulation of cyclic GMP is the coupling of the glutamate receptor to soluble guanylate cyclase. The effect of ethanol on agonist-activated activity of guanylate cyclase may contribute to the pharmacological action of ethanol in vivo.     

Inhibition of inositol 1,4,5-trisphosphate formation by cyclic GMP in cultured aortic endothelial cells of the pig.

1. In cultured endothelial cells of the pig the endothelium-derived relaxing factor (EDRF) releasing agent thrombin (2 u ml-1) caused a significant increase in basal levels of both guanosine 3':5'-cyclic monophosphate (cyclic GMP) and inositol 1,4,5-trisphosphate (IP3). This increase was time dependent, with peak levels occurring at 2 min and returning towards basal values after 5 min. 2. Pretreatment of the cells with the EDRF inhibitors haemoglobin (1 microM) or L-NG-nitro arginine (50 microM) significantly reduced the cyclic GMP response to thrombin. Both agents also resulted in significant elevations in basal levels of IP3. The IP3 response to thrombin was significantly enhanced at all time points by haemoglobin and at 5 min for L-NG-nitro arginine, when compared with the response to thrombin alone. 3. Pretreatment of the cells with either sodium nitroprusside (10 microM) or atrial natriuretic peptide (1 microM) caused a significant elevation of basal cyclic GMP levels. Although subsequent exposure to thrombin caused a further increase in cyclic GMP, which together with the rise induced by the previous two agents was significantly greater than the increase caused by thrombin alone, the incremental increase induced by thrombin was markedly less in the presence of nitroprusside or atrial natriuretic peptide. Both these agents, as well as 8-bromo cyclic GMP, resulted in a significant suppression of the IP3 response to thrombin. 4. These findings show that one mechanism for the inhibitory effect of cyclic GMP on EDRF release from endothelium may be through the inhibition of IP3 formation in response to EDRF releasing agents.     

Effects of natriuretic peptides on ventricular myocyte contraction and role of cyclic GMP signaling

Natriuretic peptides, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) act through different receptors and at different potencies to affect cardiac myocyte function. We tested the hypothesis that these three peptides would differentially reduce cardiomyocyte function through their effects on the cyclic GMP signaling pathway. Rabbit ventricular myocytes were isolated and stimulated by electrical field stimulation. Cell function was measured using a video edge detector. ANP BNP or CNP at 10⁻⁹, 10⁻⁸, 10⁻⁷ M were added to the myocytes. Intracellular cyclic GMP was determined using a radioimmunoassay in the absence or presence of ANP, BNP or CNP. All natriuretic peptides decreased myocyte contractility in a similar concentration dependent manner. Myocyte percentage shortening was significantly decreased with all peptides at 10⁻⁷ M compared with baseline (ANP from 5.4±0.4 to 3.9±0.2%; BNP from 5.0±0.2 to 3.5±0.1%; CNP from 5.6±0.3 to 4.0±0.3%). Maximum rate of shortening and relaxation were also decreased similarly and significantly. Intracellular cyclic GMP was significantly increased in myocytes treated with ANP, BNP or CNP (Baseline 1.0±0.2, ANP 2.1±0.2, BNP 2.3±0.3, CNP 2.0±0.2 pmol/10⁵ myocytes). Furthermore, inhibition of the cyclic GMP protein kinase with KT5823 caused a reversal in the functional effects of CNP. We concluded that all natriuretic peptides had similar negative effects on ventricular myocyte function and their effects were accompanied by increased cyclic GMP. Blockade the effect of CNP by a cyclic GMP protein kinase inhibitor demonstrated that effects were mediated through the cyclic GMP signaling pathway.     

Potentiation of atrial natriuretic peptide-stimulated cyclic guanosine monophosphate formation by glucocorticoids in cultured rat renal cells.

1. The effect of steroid hormones on atrial natriuretic peptide (ANP)-stimulated cyclic guanosine monophosphate (cyclic GMP) formation was studied in cultured rat renal cells. 2. ANP increased cyclic GMP formation in a dose-dependent manner, while cyclic AMP was not changed by ANP. 3. Steroid hormones did not affect basal cyclic GMP levels in cultured rat renal cells. 4. Dexamethasone at 10(-8) M increased ANP (human and rat ANP)-stimulated cyclic GMP dose-dependently in cultured rat renal cells. Cortisol, corticosterone and aldosterone at a concentration of 10(-7) M also potentiated ANP-stimulated cyclic GMP formation, although triiodothyronine, oestradiol and testosterone were ineffective. Potentiation of ANP action by these steroids seems to parallel glucocorticoid activity. 5. Dexamethasone did not affect cyclic GMP formation stimulated by sodium nitroprusside which stimulates soluble guanylate cyclase in the cytosol. Therefore, the potentiating action of dexamethasone may be mediated through the action on particulate guanylate cyclase at the plasma membrane. 6. It is suggested that the diuretic action of glucocorticoids may, at least in part, be mediated through the potentiating effect of glucocorticoids on cyclic GMP response to ANP.     

Distinct properties of atrial natriuretic factor receptor subpopulations in epithelial and fibroblast cell lines

We have characterized two atrial natriuretic factor (ANF) receptor subtypes, designated ANF-R1 and ANF-R2, in two established cell lines that express exclusively one receptor subtype. The ANF-R1 receptor is selectively expressed by the kidney epithelial cell line LLC-PK1. It is a 130-kDa protein that has a much higher affinity for the biologically active forms of ANF than for its metabolites. The binding of ANF to this subtype is potentiated by amiloride and by divalent cations. The activation of the ANF-R1 receptor leads to an accumulation of cyclic GMP that is only partially inhibited by methylene blue. The ANF-R2 receptor, which is expressed selectively by the fibroblast cell line NIH-3T3, is a 130-kDa protein composed of two disulfide-linked subunits of 64-kDa. Activation of this subtype by saturating concentrations of ANF does not appear to elicit cyclic GMP production. However, supraphysiological concentrations of ANF induce a nonsaturable accumulation of cyclic GMP with an apparent ED50 in the high micromolar range. In contrast to the ANF-R1 subtype, the stimulation of cyclic GMP production is completely abolished by methylene blue. This subtype recognizes the active forms of ANF as well as its metabolites, and the binding is insensitive to amiloride and is decreased by divalent cations. These two cell lines can serve as models for studying the differential regulatory properties of ANF-R1 and ANF-R2 subtypes. In addition, we have also characterized the two ANF receptor subtypes in rat kidney glomeruli, where they show the same structure and pharmacological characteristics as in the two model cell lines.     

The renal, cardiovascular and hormonal actions of human atrial natriuretic peptide in man; effects of indomethacin.

The renal, cardiovascular and hormonal effects of intravenous infusion of alpha-human atrial natriuretic polypeptides (alpha-hANP) at the concentrations of 0.0125, 0.025, 0.05, 0.1 microgram kg-1 min-1 for 20 min was studied in six male volunteers before and after indomethacin administration (150 mg day-1, three times daily for 3 days). Dose-dependent diuresis and natriuresis were observed in all subjects between the concentrations of 0.025 and 0.1 microgram kg-1 min-1, which were not influenced by indomethacin. Diastolic blood pressure decreased significantly (P less than 0.05) at the higher dose (0.05 microgram kg-1 min-1) of alpha-hANP, which was attenuated by indomethacin pretreatment. The plasma concentration of the immunoreactive alpha-hANP was 73.7 +/- 25 pg ml-1 on the control in subjects taking 200 mEq day-1 of sodium, and significant diuresis occurred when plasma concentration reached approximately 330.5 +/- 74.4 pg ml-1. alpha-hANP infusion caused a dose-dependent increase in cyclic GMP, no significant changes in plasma aldosterone and 18-hydroxycorticosterone, which were not influenced by indomethacin pretreatment. Plasma renin did not change in response to alpha-hANP infusion, which was significantly decreased (P less than 0.05) after indomethacin pretreatment. These results support that the renal effects of alpha-hANP may be exerted by prostaglandin-independent mechanisms. The renal effects occur at lower doses, and cardiovascular changes occur at higher doses of alpha-hANP.     

Plasma immunoreactive atrial natriuretic peptide levels after subcutaneous alpha-hANP injection in normal humans

The plasma hormone and urine effects of 100 micrograms of alpha-human atrial natriuretic peptide (alpha-hANP) given by subcutaneous (s.c.) injection, were studied in eight healthy male volunteers. A control s.c. injection was administered on a separate day, and the study was single-blind. The peak immunoreactive atrial natriuretic peptide (IR-ANP) level of 29.4 +/- 4.4 pmol/L reached at 5 min was threefold higher than on the control day. Area under the IR-ANP response curve was approximately 1/32 of that after the same dose of alpha-hANP given by intravenous (i.v.) injection. alpha-hANP s.c. injection was not associated with significant effects on plasma renin activity (PRA), plasma aldosterone, or urine electrolyte excretion. It was concluded that only a small percentage of intact alpha-hANP is absorbed into the circulation after s.c. injection. With the dose of peptide and the injection vehicle chosen, alpha-hANP had little or no biological effect.     

Enzymatic inactivation of major circulating forms of atrial and brain natriuretic peptides

We compared the enzymatic inactivation of major circulating forms of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Both ANP and BNP induced a significant increase in cyclic GMP (cGMP) formation in cultured epithelial cell line derived from porcine kidney, LLC-PK₁. The cGMP formation stimulated by ANP in LLC-PK₁ cells was significantly decreased by pre-treatment of the peptide with rat renal brush-border membranes, and the inactivation of ANP was inhibited by neutral endopeptidase inhibitors, phosphoramidon and S-thiorphan. BNP exhibited greater resistance to enzymatic inactivation than did ANP. In addition, phosphoramidon potentiated the natriuresis with a low dose (7.5 pmol min⁻¹ kg⁻¹) of ANP but not of BNP in rats. These results suggest that enzymatic degradation of natriuretic peptides is highly dependent on peptide structure, and that the affinity of BNP to neutral endopeptidase is less than that of ANP.     

Lactate Accumulation in Isolated Hypoxic Rat Ventricular Myocardium: Effect of Different Modulators of the Cyclic GMP System

Abstract: Different agents which are known to increase tissue levels of cyclic guanosine 3′5′-monophosphate (cGMP), were found to decrease the lactate accumulation induced by hypoxia in isolated, non-beating rat myocardium from the right ventricle. One μM sodium nitroprusside increased the intracellular cGMP content 4 times during hypoxic conditions, and after 5 min. of hypoxia the intracellular lactate accumulation decreased by about 20%. 0.1 μM atrial natriuretic peptide increased cGMP 10 times during hypoxic conditions and decreased the lactate accumulation by about 40%. The reduction in lactate accumulation was mimicked by 1 mM 8-Br-cGMP and by Zaprinast (10 μM), a selective inhibitor of cGMP phosphodiesterase, which reduced lactate accumulation by 60% and 45%, respectively. Glyceryl trinitrate (1 nM and 1 μM) caused a slight increase in lactate accumulation both during normooxic and hypoxic conditions, but had no effect on tissue levels of cGMP. In conclusion, the results indicate that cyclic GMP reduces lactate accumulation in hypoxic, non-beating rat heart ventricular muscle and suggests that atrial natriuretic peptide, which is released from atrial tissue, may have beneficial metabolic effects on the heart.     

Natriuretic peptide-induced cyclic GMP accumulation in adult guinea-pig cerebellar slices.

1. Second messenger responses to natriuretic peptides were studied in guinea-pig cerebellar slices by use of radioactive precursors. 2. The rank order of potency of the different natriuretic peptides in generating [3H]-guanosine 3':5'-cyclic monophosphate (cyclic GMP) was atrial natriuretic peptide (ANP) > brain natriuretic peptide (BNP) >> C-type natriuretic peptide (CNP) with EC50 values of 19.5 +/- 8.8 nM for ANP and 169 +/- 41 nM for BNP. CNP induced [3H]-cyclic GMP accumulation only at concentrations greater than 1 microM. 3. An additive response to ANP (1 microM) was observed in the presence of the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA, 10 microM) or the soluble guanylyl cyclase activator, sodium nitroprusside (SNP, 100 microM) for [3H]-cyclic GMP accumulation. 4. ANP, BNP and CNP (all at 1 microM) failed to alter significantly either basal-, forskolin- (10 microM), isoprenaline- (100 microM), or NECA- (10 microM) induced [3H]-cyclic AMP generation. Natriuretic peptides also did not change the [3H]-cyclic AMP steady-state reached after 10 min of treatment with 10 microM forskolin. 5. Natriuretic peptides failed to elicit significant accumulation of [3H]-inositol phosphates at concentrations up to 10 microM. 6. These data are consistent with the presence of ANPA, rather than ANPB or clearance receptors (C-receptors), linked to second messenger cascades in guinea-pig cerebellar slices.     

Structure-activity relationships of alpha-human atrial natriuretic peptide

The spasmolytic activity of synthetic alpha-human atrial natriuretic peptide (alpha-hANP) and its related peptides was determined in vitro using the chick rectum and the rat aorta. Natriuretic activity was also measured in the anesthetized rat, alpha-hANP-(7-28), with the NH2-terminal hexapeptide truncated, had greater spasmolytic and natriuretic effect than did alpha-hANP-(1-28). These responses were reduced by truncation of the COOH-terminal residues. alpha-hANP-(7-23), the cyclic structure of alpha-hANP-(1-28), exhibited weak aortic relaxation and natriuretic activities. However, alpha-hANP-(7-23) produced a greater relaxation than did alpha-hANP-(1-28) in the chick rectum. Elimination of Gly at position 9 reduced the spasmolytic and natriuretic activity. Substitution of amino acid residues at position 8, 12 and 13 changed these activities. Analogues containing the ethylene linkage instead of the disulphide bond had weak biological activity. These results indicate that the size of the 17-amino acid ring and the COOH-terminal residues of alpha-hANP are important for the expression of spasmolytic and natriuretic activity.     

Cross-tolerance to L-arginine-dependent guanylate cyclase activators in nitrate-tolerant LLC-PK1 kidney epithelial cells

Recently, it was shown that in LLC-PK1 kidney epithelial cells hormones such as vasopressin or oxytocin increase cyclic GMP in a receptor-mediated and L-arginine-dependent manner. In the present study, the possible existence of cross-tolerance to vasopressin and oxytocin was investigated in nitrate-tolerant LLC-PK1 cells. Pretreatment with 1 mM glyceryl trinitrate for 3 h decreased cyclic GMP stimulation by 1 microM vasopressin and 1 microM oxytocin by 49% and 54%, respectively. Under the same conditions, cyclic GMP stimulation at 1 microM sodium nitroprusside was diminished by 56% whereas the cyclic GMP response to 100 microM glyceryl trinitrate was virtually abolished. Our results demonstrate that a substantial degree of cross-tolerance to L-arginine-dependent guanylate cyclase activators occurs in nitrate-pretreated nonvascular cells which may be due to glyceryl trinitrate-induced desensitization of soluble guanylate cyclase.